The long-term program goal is to understand why elderly hearts of humans and animal models are more prone to irreversible ischemic injury. In conjunction with other projects of this PPG that are aimed at characterizing specific age-and ischemia-dependent changes in cardiomyocytes due to increases in mitochondrial-generated radical species, this project focuses on increased susceptibility of the cardiomyocytes to apoptosis as a contributing mechanism. Oxyradicals modify glutathione (GSH) and critical sulfhydryl groups on proteins that are vital for many cell functions including energy production, ion transport, gene regulation, membrane integrity, and regulation of the balance between cell survival and controlled cell death (apoptosis). The operating hypothesis is that the net effect of cardiovascular damage associated with ischemia-reperfusion injuries is likely exacerbated by age-related changes in homeostatic regulation of protein thiol-disulfide status. Therefore this project focuses on the relationship between oxidative-stress-induced changes in glutaredoxin-mediated redox regulation of reversible S-glutathionylation of critical proteins and the susceptibility of the cells to undergo apoptosis. Using the primary animal model Fischer 344 rats during the current funding period, glutaredoxin (GRxl) content and activity were found to be decreased in cytosol, and the net glutaredoxin activity (GRX1 + GRx2) appeared to be decreased also in mitochondria of heart cells from elderly rats compared to young adult rats. In contrast thioredoxin and thioredoxin reductase were unchanged in cytosol and mitochondria with age. Accordingly the focus for continuation of this project is on the glutaredoxin system and its role in regulation of S-glutathionylation of cellular proteins (protein-SSG). The mechanisms of changes in cytosolic and mitochondrial glutaredoxin activities will be investigated, and the role of glutaredoxin in regulating the activities of key mediators of apoptotic signaling in cardiomyocytes of young adult and elderly rat hearts will be studied to ascertain how this control network contributes to increased myocardial damage after ischemicreperfusion injury. In particular, changes in the protein-SSG status of key mediators of apoptotic regulation in the mitochondria and the cytosol will be studied as a function of age and ischemia / reperfusion, and correlated with the content and distribution of glutaredoxin in natural hearts and in hearts in which the content and/or form of glutaredoxin have been manipulated by in vivo gene transfer. The animals with genetically altered contents of heart glutaredoxin will be studied collaboratively by all of the projects. These studies will enhance our general understanding of the role of glutaredoxin in apoptotic regulation while addressing potential mechanisms for age-dependent predisposition of hearts to irreversible injury.